CN118064393A - Ketone reductase mutant and application thereof - Google Patents
Ketone reductase mutant and application thereof Download PDFInfo
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- CN118064393A CN118064393A CN202410190729.3A CN202410190729A CN118064393A CN 118064393 A CN118064393 A CN 118064393A CN 202410190729 A CN202410190729 A CN 202410190729A CN 118064393 A CN118064393 A CN 118064393A
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- ketoreductase
- amino acid
- acid sequence
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Abstract
The invention relates to a ketoreductase mutant and application thereof, wherein the ketoreductase mutant is obtained by mutating an amino acid sequence shown in SEQ ID NO.1, and the mutating comprises at least one of the following mutating sites: mutation from Y to F or M at position 157, from K to M or A at position 161, from P to A or W at position 187, and from T to L or A at position 192. The ketoreductase mutant can be applied to catalyzing ketone compounds to prepare chiral alcohol compounds, and has high catalytic activity and good stability. Compared with a chemical synthesis method, the catalytic reaction has the advantages of simple operation, mild reaction conditions, high reaction selectivity, low preparation cost and good application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a ketoreductase mutant and application thereof.
Background
Chiral alcohols are widely used as important intermediates in the synthesis of chiral drugs and other chiral fine chemicals. Many chiral drugs contain one or more chiral centers and there are significant differences in pharmacological activity, metabolic processes, metabolic rates, and toxicity of different chiral drugs. Chiral alcohols are produced in various ways, such as lower theoretical yields and higher costs by kinetic resolution. Chiral oxazaborolidine hydroboration and chiral transition metal may be also used in asymmetric catalytic hydrogenation to synthesize chiral alcohol, and the reaction condition is relatively severe, such as high temperature, high pressure, etc. and the problem of transition metal and other toxic reagent residue is also involved.
Ketoreductase stereoselectively reduces ketone to alcohol, and ketoreductase is used as a catalyst to replace a traditional chemical catalyst, so that the ketoreductase has the advantages of mild reaction, less environmental pollution and the like, and great attention is paid to the field of pharmacy.
Catalytic synthesis of chiral alcohols using ketoreductase is a very potential catalytic approach, but this approach also has several problems, including lack of stability of ketoreductase itself under the production reaction conditions, product inhibition, low catalytic activity of the enzyme, low selectivity for catalytic reduction of substrates, low water solubility of many substrates of interest, etc. Therefore, in many specific ketosubstrates, the catalytic effect of the existing ketoreductase is poor in the process of catalytically reducing the ketoreductase into chiral alcohol, so that a suitable ketoreductase needs to be found, or the activity and the specificity of the known ketoreductase are improved through rational design, so that the catalytic reduction of the ketoreductase into alcohol with a specific configuration is promoted.
For example, vitamin Bei Gelong (Vibegron) is a beta-3 adrenergic agonist for the treatment of urge incontinence, urgency and frequency symptoms in patients with overactive bladder (OAB). For the compound of formula I-4 of intermediate vitamin Bei Gelong (structure shown in scheme 1 below), it can be obtained by the catalytic reduction of the compound of formula I-3 by a ketoreductase, but for the unnatural substrate of the compound of formula I-3, the catalytic reduction effect of the existing ketoreductase for the enzyme is lower due to the specificity of the enzyme, or other defects such as poor stability, difficult availability, etc. exist. For example, prior art WO2014150639A1 reports reaction 1 using a ketoreductase to catalyze the reduction of a compound of formula I-3 to a compound of formula I-4, but the ketoreductase referred to in this prior art is a wet gel immobilized ketoreductase (which is prepared by first dissolving the ketoreductase in an aqueous solution containing disodium hydrogen phosphate and NADP, then adding polymethacrylate, DIAION TM HP2MG, and finally pouring out the solution, and washing the wet gel with dipotassium hydrogen phosphate to obtain an immobilized ketoreductase) and the purpose of gel immobilizing the ketoreductase is to overcome the disadvantage of poor stability of the ketoreductase in an organic solvent (this reason is mentioned in another prior art WO 2014/150633). Wet gel immobilized ketoreductase needs to be prepared in advance, is inconvenient to use,
Thus, there is a need for a ketoreductase enzyme that is less useful, simple to use, has good activity and good selectivity for the reduction of compounds of formula I-3.
Disclosure of Invention
One aspect of the invention provides a ketoreductase mutant capable of catalyzing ketone compounds to prepare chiral alcohol compounds, which has high activity and good selectivity, and can be directly catalyzed by thallus wet cells containing the ketoreductase mutant. In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
A ketoreductase mutant, the amino acid sequence of which is obtained by mutating the amino acid sequence shown in SEQ ID NO.1, wherein the mutation at least comprises one of the following mutation sites: the 157 th position is mutated from Y to F or M, the 161 th position is mutated from K to M or A, the 187 th position is mutated from P to A or W, the 192 th position is mutated from T to L or A, or the ketoreductase mutant has a mutation site in the mutated amino acid sequence, and the amino acid sequence has more than 85% homology with the mutated amino acid sequence.
In another preferred embodiment, the amino acid sequence of the ketoreductase mutant is shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8; or the amino acid sequence of the ketoreductase mutant has more than 85 percent of homology with the amino acid sequence shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8.
In another preferred embodiment, the amino acid sequence of the ketoreductase mutant is shown as SEQ ID NO.4 or SEQ ID NO. 5. In another more preferred embodiment, the amino acid sequence of the ketoreductase mutant is shown in SEQ ID NO. 5.
In another aspect, the invention provides a polynucleotide encoding a ketoreductase mutant as described above.
In another preferred embodiment, the polynucleotide has a sequence as shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO.14, or the polynucleotide has a nucleotide sequence having more than 85% homology with the nucleotide sequence as shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO. 14.
In another preferred embodiment, the nucleotide sequence of the polynucleotide is shown as SEQ ID NO.10 or SEQ ID NO. 11.
In another aspect, the present invention provides a recombinant expression vector comprising the polynucleotide described above.
In another aspect, the invention provides a host cell comprising the recombinant expression vector described above.
The invention also provides the application of the ketoreductase mutant in preparing chiral alcohol compounds by catalyzing ketone compounds,
The structural formula of the ketone compound isWherein R 1 is selected from phenyl, R 2 is selected from C 1～C8 alkyl, wherein said phenyl and said C 1～C8 alkyl are optionally further substituted with one or more substituents selected from hydroxy, amino, halo, nitro, cyano, carboxy, C 1～C8 alkyl, C 1～C8 alkoxy, C 5～C10 cycloalkyl, C 2～C6 ester, C 2～C8 alkenyl, C 2～C8 alkynyl, C 4～C11 heterocyclyl, C 5～C10 aryl, C 5～C10 heteroaryl,
The alcohol compound isWherein R 1 and R 2 are as defined above.
In another preferred embodiment, the ketone compound is a compound of formula I,The alcohol compound is a compound of formula II:/>
In yet another aspect, the present invention provides a process for the preparation of a compound of formula II, comprising the steps of:
Reducing a compound shown in a formula I in the presence of ketoreductase to obtain a compound shown in a formula II, wherein the reaction formula is as follows:
wherein the amino acid sequence of the ketoreductase is shown as SEQ ID NO. 5.
In another preferred embodiment, the ketoreductase is in free form, immobilized form or in bacterial form.
In another preferred embodiment, the ketoreductase is in the form of a bacterial cell. In another preferred embodiment, the ketoreductase in the form of a bacterial cell is obtained by centrifugation.
In another preferred embodiment, the ratio by weight of ketoreductase in the form of cells obtained by centrifugation to the substrate in the reaction system is 5% to 40%, more preferably 10% to 30%.
In another preferred embodiment, the ratio by weight/volume of the ketoreductase in the form of cells obtained by centrifugation to the reaction solvent in the reaction system is 5g/L to 50g/L, more preferably 10g/L to 30g/L.
In another preferred embodiment, the centrifugation to obtain the ketoreductase in bacterial form comprises the steps of: centrifuging at 10000-15000 rpm for 8-15 min, and discarding supernatant to obtain cell precipitate, namely thallus type ketoreductase.
In another preferred embodiment, in the process for the preparation of the compounds of the formula II, the coenzyme is also present in the reaction system.
In another preferred embodiment, the coenzyme is selected from NADP+, NADP salts or NADPH, more preferably NADP salts.
In another preferred embodiment, the amount of NADP+, NADP salt or NADPH to substrate is 0.01-1% (w/w), more preferably 0.01-0.1% (w/w).
In another preferred embodiment, in the process for preparing the compounds of the formula II, enzymes for the regeneration of the coenzyme are also present in the reaction system.
In another preferred embodiment, the enzyme used for coenzyme regeneration is selected from the group consisting of alcohol dehydrogenase, glucose dehydrogenase, formate dehydrogenase, or a combination thereof.
In another preferred embodiment, in the process for the preparation of the compounds of the formula II, co-substrates for the regeneration of the coenzyme are also present in the reaction system.
In another preferred embodiment, the co-substrate is selected from isopropanol, glucose, formic acid, or a combination thereof. In another preferred embodiment, the concentration of the co-substrate in the reaction system is 5 to 30% by mass.
In another preferred embodiment, the reaction temperature of the reaction system in the process for preparing the compounds of the formula II is from 20 to 50℃and more preferably from 30 to 40 ℃.
In another preferred embodiment, the pH of the reaction system in the process for preparing the compounds of the formula II is from 6 to 9, more preferably from 7 to 8.
In another preferred embodiment, the compound of formula II is prepared by a process wherein the reaction system is water and an organic solvent selected from dimethyl sulfoxide, methanol, ethanol, isopropanol, acetonitrile, toluene, acetone, or a combination thereof. In another preferred example, the volume ratio of the organic solvent to the water is 1:0.5-1:2.
The ketoreductase mutant is obtained by mutating the ketoreductase shown in SEQ ID NO.1 by a site-directed mutation method, so that the amino acid sequence of the ketoreductase mutant is changed, the change of the structure and the function of the protein is realized, and the ketoreductase with the mutation site is obtained by a directional screening method. For the reduction of the ketone compounds, the enzyme selectivity of the ketoreductase mutant is improved by multiple times compared with that of the wild ketoreductase, the enzyme activity is also greatly improved, and the cost in the industrial production of chiral alcohol is greatly reduced when the ketoreductase mutant is used for the production of chiral alcohol.
Drawings
FIG. 1 is a diagram of a recombinant expression vector for ketoreductase of the invention.
FIG. 2 shows the results of polyacrylamide gel electrophoresis of different ketoreductases, from left to right, the proteases Marker, BL21 (DE 3) -iii (with the amino acid sequence SEQ ID NO. 5), BL21 (DE 3) -ii (with the amino acid sequence SEQ ID NO. 4), BL21 (DE 3) -i (with the amino acid sequence SEQ ID NO. 3), wild-type protease (with the amino acid sequence SEQ ID NO. 1), and blank, respectively.
FIG. 3 shows the results of polyacrylamide gel electrophoresis of different ketoreductases, from left to right, the proteases Marker, BL21 (DE 3) -iv (with the amino acid sequence SEQ ID NO. 6), BL21 (DE 3) -v (with the amino acid sequence SEQ ID NO. 7), BL21 (DE 3) -vi (with the amino acid sequence SEQ ID NO. 8) and wild-type proteases, respectively.
FIG. 4 is a liquid chromatography of the reaction solution of example 6, in which the substrate (RT: 12.228 minutes) and the product (RT: 13.617 minutes) were used.
Detailed Description
Ketoreductase derived from Rhodococcus erythropolis can selectively catalyze the reduction of carbonyl groups, but is less active and less selective on the above-described compounds of formula I. The inventors of the present invention increased the activity and selectivity of the ketoreductase derived from Rhodococcus erythropolis by a rational design approach. Mutant sites are introduced into aminotransferase from Rhodococcus erythropolis by means of whole plasmid PCR, and activity and stability of the mutants are detected, so that mutants with improved activity and stability are selected.
The ketoreductase mutant polynucleotide provided by the invention is derived from a Rhodococcus erythropolis wild type polynucleotide. The amino acid sequence of the wild polynucleotide is shown as SEQ ID NO.1, and the polynucleotide sequence after codon optimization for escherichia coli is shown as SEQ ID NO. 2. Wherein "wild type" refers to a form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence that is present in an organism, can be isolated from a natural source and is not intentionally modified by human manipulation. The enzyme obtained after the expression of the polynucleotides has low catalytic activity on certain substrates and poor thermal stability.
The amino acid sequence of the ketoreductase from Rhodococcus erythropolis is shown in SEQ ID NO. 1.
CDNA of Rhodococcus erythropolis ketoreductase, and polynucleotide sequence of the cDNA after codon optimization for colibacillus are shown in SEQ ID NO. 2.
The ketoreductase sequence is derived from GenBank: BAF43657.1, homologous modeling is carried out by taking 6ci9 as a template (Identity: 64.06%) to obtain a ketoreductase protein three-dimensional structure, then the combination simulation of a substrate of the formula I and the ketoreductase protein three-dimensional structure is carried out through Autodock, and finally amino acid possibly related to substrate combination is selected as mutant amino acid through Pymol analysis. According to the analysis result of Pymol, a plurality of pairs of site-directed mutagenesis (Y157F/M; K161M/A; P187A/W; T192L/A) primers are designed, and a site-directed mutagenesis means is utilized to obtain mutant plasmids with target polynucleotides by taking pET-28a, pET-dute1 or pRSF-dute1 as expression vectors. Wherein, site-directed mutagenesis: refers to the introduction of desired changes (typically changes characterizing the advantageous direction) into a DNA fragment of interest (which may be a polynucleotide set or a plasmid) by Polymerase Chain Reaction (PCR) or the like, including addition, deletion, point mutation, etc. The site-directed mutagenesis can rapidly and efficiently improve the properties and characterization of target proteins expressed by DNA, and is a very useful means in polynucleotide research work.
The ketoreductase mutant provided by the application is an amino acid sequence obtained by mutating the amino acid sequence shown in SEQ ID NO.1, wherein the mutation at least comprises one of the following mutation sites: the 157 th position is mutated from Y to F or M (Y157F or M), the 161 th position is mutated from K to M or A (K161M or A), the 187 th position is mutated from P to A or W (P187A or W), the 192 th position is mutated from T to L or A (T192L or A), or the amino acid sequence of the ketoreductase mutant has a mutation site in the mutated amino acid sequence and has an amino acid sequence with more than 85% homology with the mutated amino acid sequence. Wherein, "homology" refers to the identity between two amino acid sequences. The sequences defined by the different degrees of homology according to the application must also have an improved ketoreductase activity at the same time. Those skilled in the art can, under the teachings of the present disclosure, obtain amino acid sequences of ketoreductase mutants that have mutation sites in the mutated amino acid sequences described above and that have more than 85% homology with the mutated amino acid sequences.
In a specific embodiment of the present invention, the ketoreductase mutant comprises mutation sites Y157F (mutation of Y to F at position 157) and K161M (mutation of K to M at position 161), the amino acid sequence of which is shown in SEQ ID NO.3, or the ketoreductase mutant comprises mutation sites Y157F and K161M, and the amino acid sequence has more than 85% homology with the amino acid sequence shown in SEQ ID NO. 3. In a specific embodiment of the present invention, the above ketoreductase mutant comprises mutation sites Y157F, K161M and P187W (the 187 th site is mutated from P to W) and the amino acid sequence thereof is shown in SEQ ID NO.4, or the above ketoreductase mutant comprises mutation sites Y157F, K161M and P187W and the amino acid sequence thereof has a homology of 85% or more with the amino acid sequence shown in SEQ ID NO. 4. In one embodiment of the present invention, the ketoreductase mutant comprises mutation sites Y157F, K161M, P187W and T192A (the 192 st site is mutated from T to A) with the amino acid sequence shown in SEQ ID NO.5, or the ketoreductase mutant comprises mutation sites Y157F, K161M, P187W and T192A with the amino acid sequence shown in SEQ ID NO.5 with the amino acid sequence with more than 85% homology. In a specific embodiment of the present invention, the ketoreductase mutant comprises mutation sites Y157M (mutation of Y to M at position 157) and K161A (mutation of K to A at position 161), the amino acid sequence of which is shown in SEQ ID NO.6, or the ketoreductase mutant comprises mutation sites Y157M and K161A, and has an amino acid sequence with homology of 85% or more with the amino acid sequence shown in SEQ ID NO. 6. In a specific embodiment of the present invention, the above ketoreductase mutant comprises mutation sites Y157M, K161A and P187A (the 187 th site is mutated from P to A), the amino acid sequence of which is shown in SEQ ID NO.7, or the above ketoreductase mutant comprises mutation sites Y157M, K161A and P187A, and an amino acid sequence having homology of 85% or more with the amino acid sequence shown in SEQ ID NO. 7. In one embodiment of the present invention, the ketoreductase mutant comprises mutation sites Y157M, K161A, P187A and T192L (the 192 st position is mutated from T to L) and the amino acid sequence is shown as SEQ ID NO.8, or the ketoreductase mutant comprises mutation sites Y157M, K161A, P187A and T192L and the amino acid sequence has more than 85% homology with the amino acid sequence shown as SEQ ID NO. 8.
The ketoreductase mutant is obtained by mutating the ketoreductase shown in SEQ ID NO.1 by a site-directed mutation method so as to change the amino acid sequence of the ketoreductase mutant, realize the change of the structure and the function of the protein and obtain the ketoreductase with the mutation site by a directional screening method. For the reduction of the ketone compounds, the ketoreductase mutant has the advantage of greatly improving the enzyme selectivity. The ee value of the product obtained by reducing the compound shown in the formula I by using the ketoreductase mutant (particularly the ketoreductase mutant with the amino acid sequence shown in SEQ ID NO. 5) is greatly improved compared with the ee value of the product obtained by using wild ketoreductase under the same condition, and the enzyme activity is correspondingly improved, so that the cost in the industrial production of chiral alcohol is greatly reduced.
The present invention provides polynucleotides encoding the above ketoreductase mutants. The invention mutates the polynucleotide of wild ketoreductase by rational design (site-directed mutagenesis or other methods to change individual amino acids in protein molecules), overlap extension PCR, seamless cloning and other methods to obtain the target polynucleotide of the ketoreductase mutant. In one embodiment of the invention, the nucleotide sequence of the polynucleotide is shown as SEQ ID NO.9 (which encodes the amino acid sequence shown as SEQ ID NO. 3), or the nucleotide sequence of the polynucleotide has more than 85% homology with the sequence shown as SEQ ID NO. 9. In one embodiment of the invention, the nucleotide sequence of the polynucleotide is shown as SEQ ID NO.10 (which encodes the amino acid sequence shown as SEQ ID NO. 4), or the nucleotide sequence of the polynucleotide has more than 85% homology with the sequence shown as SEQ ID NO. 10. In one embodiment of the invention, the nucleotide sequence of the polynucleotide is shown as SEQ ID NO.11 (which encodes the amino acid sequence shown as SEQ ID NO. 5), or the nucleotide sequence of the polynucleotide has more than 85% homology with the sequence shown as SEQ ID NO. 11. In one embodiment of the invention, the nucleotide sequence of the polynucleotide is shown as SEQ ID NO.12 (which encodes the amino acid sequence shown as SEQ ID NO. 6), or the nucleotide sequence of the polynucleotide has more than 85% homology with the sequence shown as SEQ ID NO. 12. In one embodiment of the invention, the nucleotide sequence of the polynucleotide is shown as SEQ ID NO.13 (which encodes the amino acid sequence shown as SEQ ID NO. 7), or the nucleotide sequence of the polynucleotide has more than 85% homology with the sequence shown as SEQ ID NO. 13. In one embodiment of the invention, the nucleotide sequence of the polynucleotide is shown as SEQ ID NO.14 (which encodes the amino acid sequence shown as SEQ ID NO. 8), or the nucleotide sequence of the polynucleotide has more than 85% homology with the sequence shown as SEQ ID NO. 14. Wherein "homology" refers to the identity between two nucleotide sequences.
As used herein, "above 85% means 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
The amino acid sequences of the above nucleoside phosphorylases and their corresponding polynucleotide sequences are shown in table 1.
Table 1.
The ketoreductase obtained by the polynucleotide codes improves the enzyme activity and the enzyme stability. The industrial production efficiency of chiral alcohol is higher, and the cost is lower.
The invention provides vectors comprising polynucleotides encoding the ketoreductase mutants of the invention. The polynucleotide is positioned in a proper position of the recombinant expression vector, so that the polynucleotide can be correctly and smoothly copied, transcribed or expressed. In order to meet the requirements of recombinant operation, the two ends of the polynucleotide sequence can be added with the enzyme cutting sites of proper restriction endonuclease or additionally added with a start codon, a stop codon and the like. The vector used for constructing the recombinant expression vector can be a prokaryotic expression vector or a eukaryotic expression vector. In the present invention, the recombinant expression vector includes, but is not limited to, pET-28a, pET-dute1 or pRSF-dute1.
The host cell provided by the invention is used for producing the ketoreductase mutant, and comprises the vector. In the present invention, the host cell includes, but is not limited to, E.coli MG1655 or E.coli BL21 (DE 3) pLysS.
The ketoreductase mutants described above may be prepared by culturing the host cells by fermentation. For example, the above ketoreductase mutants are industrially produced under the conditions of tank fermentation. The fermentation conditions of the production tank are preferably as follows: DO is above 20% and the temperature is 20 ℃.
In a preferred embodiment of the invention, when the ketoreductase group is mutated, a seamless cloning mode is used, and primers on the pET28a plasmid are respectively positioned at the upstream and downstream of the ketoreductase polynucleotide. The mutation site is provided with a primer with 15bp homologous arms at two ends, and the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 53-60℃for 15s and extension at 72℃for 50s for 30 cycles; the extension was continued at 72℃for 10min and cooled to 4 ℃. The PCR amplified fragment is connected with pET28a plasmid vector by using a seamless cloning kit, the connected vector is transferred into escherichia coli BL21 (DE 3) to establish ketoreductase polynucleotide mutant, and the extension mutated ketoreductase is expressed by using escherichia coli BL21 (DE 3) as a host and pET28a plasmid as a vector.
The ketoreductase mutant provided by the invention can catalyze carbonyl compounds to prepare chiral alcohol.
The ketone reduction mutant of the invention requires the participation of a coenzyme, such as NADH (reduced coenzyme I) or NADPH (reduced coenzyme II), in the process of reducing ketone compounds, and the combined use of the coenzyme and the mutant has high stereoselectivity and chemical selectivity on the reduction of ketone substrates compared with a chemical method, and is catalyzed in situ in one step. Wherein NAD (P) H is recycled during the reaction, and the recycling method can be realized through the process of converting isopropanol into acetone by alcohol dehydrogenase, or through the process of converting glucose into gluconic acid by glucose dehydrogenase (glucose dehydrogenase, GDH), or through the process of converting formic acid into carbon dioxide by formate dehydrogenase (formate dehydrogenase, FDH). In the reaction system for preparing chiral alcohol by catalyzing carbonyl compounds by using the ketoreductase mutant provided by the invention, NADPH, glucose and glucose dehydrogenase are preferably added.
In the description of the present invention, the term "room temperature" or "normal temperature" means a temperature of 4-40 ℃, preferably 25.+ -. 5 ℃.
In the description of the present invention, "C 1-C8 alkyl" when used as a group or part of a group refers to straight or branched aliphatic hydrocarbon groups comprising 1 to 8 carbons. Preferably a C 1–C6 alkyl group, more preferably a C 1–C4 alkyl group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, and the like. Alkyl groups may be substituted or unsubstituted.
In the description of the present invention, "C 1～C8 alkoxy" refers to a group of C 1～C8 alkyl-O-, wherein alkyl is as defined above. For example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy and the like.
In the description of the present invention, "C 5～C10 cycloalkyl" refers to saturated or partially saturated monocyclic, fused, bridged and spiro carbocycles. More preferably C 5-C8 cycloalkyl, most preferably C 5-C6 cycloalkyl. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like, with cyclopentyl, cyclohexyl being preferred. Cycloalkyl groups may be optionally substituted or unsubstituted.
In the description of the present invention, "heterocyclyl" refers to a non-aromatic heterocyclic group in which one or more of the ring-forming atoms is a heteroatom, such as oxygen, nitrogen, sulfur, and the like, including monocyclic, polycyclic, fused, bridged and spiro rings. Preferably a 4-11 membered heterocyclic ring, which may contain 1,2 or 3 atoms selected from nitrogen, oxygen and/or sulphur. Examples of "heterocyclyl" include, but are not limited to, morpholinyl, oxetanyl, thiomorpholinyl, tetrahydropyranyl, 1-dioxo-thiomorpholinyl, piperidinyl, 2-oxo-piperidinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazin-2-one, 8-oxa-3-aza-bicyclo [3.2.1] octyl, piperazinyl.
In the description of the present invention, "aryl" refers to a carbocyclic aromatic system containing one or two rings, wherein the rings may be linked together in a fused manner. The term "aryl" includes monocyclic or bicyclic aryl groups, e.g., phenyl, naphthyl, tetrahydronaphthyl aromatic groups. Aryl groups may be substituted or unsubstituted.
In the description of the present invention, "heteroaryl" refers to an aromatic 5-to 6-membered monocyclic or 8-to 10-membered bicyclic ring, which may contain 1 to 4 atoms selected from nitrogen, oxygen and/or sulfur. For example, furyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, isoxazolyl, oxazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, benzothienyl, benzimidazolyl, indolyl, isoindolyl, quinolinyl, indazolyl, and the like, and heteroaryl may be substituted or unsubstituted.
In the description of the present invention, carboxyl refers to-COOH.
In the description of the present invention, "C 2～C6 ester group" means an ester group having 2 to 6 carbons.
In the description of the present invention, "C 2～C6 alkenyl" refers to a group containing 2 to 6 carbons and having at least one olefinic bond.
In the description of the present invention, "C 2～C6 alkynyl" refers to groups containing 2 to 6 carbons and having at least one alkyne bond.
Compared with the prior art, the invention has the following advantages and effects:
1. The ketoreductase mutant (particularly the ketoreductase mutant with the amino acid sequence shown as SEQ ID NO. 5) provided by the invention ensures that the conversion rate of the compounds shown in the formulas I and A is more than 99% in 20 h; under the same conditions, the wild-type enzyme results in less than 15% conversion of such substrate; furthermore, the ee value of the chiral alcohol product obtained by reducing the compound of formula I with the enzyme mutants of the invention, in particular the enzyme mutants shown in SEQ ID No.5, can reach 99.5% respectively, whereas the ee value of the chiral alcohol product obtained under the same conditions using the wild-type enzyme is 0. Therefore, the catalytic activity of the ketoreductase mutant provided by the invention on carbonyl substrates is greatly improved compared with that of wild ketoreductase. The ketone reductase provided by the invention is used for catalyzing carbonyl substrates to synthesize chiral alcohol compounds, so that the green chemical synthesis of chiral alcohol compounds shown in a formula II is realized.
2. The ketoreductase mutant provided by the invention has high catalytic activity and good stereoselectivity, and can be applied to biosynthesis of chiral alcohols (especially compounds shown in a formula II); compared with the chemical synthesis method, the catalytic reduction reaction is simple and mild, the reaction selectivity is high, the preparation cost is low, and the method has good application prospect.
3. In the catalytic substrate reduction process of the ketoreductase mutant provided by the invention, wet whole cells containing the ketoreductase mutant can be directly added into a reaction system to perform catalytic reduction on a ketone compound substrate, and after thallus is not required to be crushed to obtain a crushing liquid, the crushing liquid is added into the reaction system.
4. The ketoreductase mutant provided by the invention has high activity and small dosage, and can basically react the substrate completely (92% or more of the substrate has been converted) at room temperature for 24-36 hours by using only wet whole cells accounting for about 10-30% of the weight of the product.
The present invention will be described in further detail with reference to the following examples, which are illustrative of the present invention and are not intended to limit the present invention thereto. The experimental methods used in the following examples are conventional methods unless otherwise specified. The raw materials, reagents and the like used in the following examples are all commercially available unless otherwise specified.
The composition of the LB liquid (per liter) used in the following examples was 10 g.+ -. 0.1 for peptone; 5 g+ -0.1 of yeast extract; naCl 10g + -0.1.
The glucose dehydrogenase used in the following examples was Shang Ke, applied biosciences (Shanghai), inc., ES-GDH-109, 40U/mg.
The compounds of formula I used in the examples below are commercially available and can also be prepared according to the methods disclosed in the prior art.
EXAMPLE 1 establishment of wild ketoreductase Polynucleotide engineering bacteria
A full polynucleotide fragment (the nucleotide sequence is shown as SEQ ID NO. 2) is artificially synthesized after sequence optimization is carried out according to Rhodococcus erythropolis ketoreductase wild type polynucleotide sequence (GenBank: BAF 43657.1) recorded by NCBI, the polynucleotide is inserted into pET-28a plasmid (figure 1) through NdeI and XhoI endonucleases by a polynucleotide synthesis company, the linked vector is transferred into escherichia coli BL21 (DE 3) to establish wild type ketoreductase polynucleotide engineering bacteria, and sequencing verification is carried out after kanamycin resistance screening.
EXAMPLE 2 design of ketoreductase mutants
Carrying out homologous modeling by taking 6ci9 as a template (Identity: 64.06%) to obtain a three-dimensional structure of the ketoreductase protein, then carrying out binding simulation of a substrate of the formula I and the three-dimensional structure of the ketoreductase protein through AutoDock, and finally selecting amino acids possibly related to substrate binding as mutant amino acids through Pymol analysis.
According to the above-mentioned Pymol analysis result, mutation is made at least 1 site in the amino acid sequence of the wild-type ketoreductase shown in SEQ ID NO. 1: 157 th, 161 th, 187 th, 192 th. And the 157 th position is mutated from Y to F or M, the 161 th position is mutated from K to M or A, the 187 th position is mutated from P to A or W, and the 192 th position is mutated from T to L or A. Finally, the amino acid sequence containing the following combined mutation sites is obtained through genetic engineering: (i) Y157F, K161M (corresponding to the amino acid sequence shown in SEQ ID NO. 3); (ii) Y157F, K161M, P187W, (corresponding to the amino acid sequence shown in SEQ ID NO. 4); (iii) Y157F, K161M, P187W, T192A (corresponding to the amino acid sequence shown in SEQ ID NO. 5); (iv) Y157M, K161A (corresponding to the amino acid sequence shown in SEQ ID NO. 6); (v) Y157M, K161A, P187A (corresponding to the amino acid sequence shown in SEQ ID NO. 7); (vi) Y157M, K161A, P187A, T192L (corresponding to the amino acid sequence shown in SEQ ID NO. 8) and high activity and high selectivity mutants were selected therefrom.
EXAMPLE 3 construction of recombinant expression vectors
TABLE 2 sequences corresponding to the mutant primers
The expression vector pET28a (+) (see FIG. 1) was digested with restriction enzymes NdeI and XhoI, respectively, in the following manner: pET28a (+) 84uL (about 30. Mu.g), ndeII. Mu. L, xhoI. Mu.L, was digested at 37℃for 1.5h and subjected to agarose gel electrophoresis. The target band was located at 0.76kb, and the digested fragments were recovered using a DNA recovery kit.
The coding sequence of ketoreductase containing the combined mutation site (i) takes the polynucleotide sequence shown in the synthesized SEQ ID NO.2 as a template, and uses a primer P 1/P2,P3/P4 to carry out PCR amplification, and a band of about 0.76k is separated after agarose gel electrophoresis, namely the nucleotide sequence shown in SEQ ID NO. 9; the ketoreductase comprising the combined mutation site (ii) is subjected to PCR amplification by taking the synthesized polynucleotide shown as SEQ ID NO.9 as a template, and by using a primer P 5/P6, and a band of about 0.76k, namely a nucleotide sequence shown as SEQ ID NO.10, is separated after agarose gel electrophoresis; the ketoreductase comprising the combined mutation site (iii) was PCR amplified using primer P 7/P8 using the polynucleotide sequence shown in SEQ ID NO.10 as a template, and a band of about 0.76k, i.e., the nucleotide sequence shown in SEQ ID NO.11, was separated after agarose gel electrophoresis. The coding sequence of ketoreductase comprising the combined mutation site (iv) is subjected to PCR amplification by using the synthesized polynucleotide sequence shown as SEQ ID NO.2 as a template and using a primer P 9/P10,P11/P12, and a band of about 0.76k, namely the nucleotide sequence shown as SEQ ID NO.12, is separated after agarose gel electrophoresis; the ketoreductase comprising the combined mutation site (iv) uses the synthesized polynucleotide shown as SEQ ID NO.12 as a template for PCR amplification, and primer P 13/P14 is used for PCR amplification, and a band of about 0.76k is separated after agarose gel electrophoresis, namely the nucleotide sequence shown as SEQ ID NO. 13; the ketoreductase comprising the combined mutation site (vi) takes the polynucleotide sequence shown in the synthetic SEQ ID NO.13 as a template, the primer P 15/P16 is used for PCR amplification, a band of about 0.76k is separated after agarose gel electrophoresis, namely the nucleotide sequence shown in SEQ ID NO.14, and six groups of PCR amplified products are recovered by a DNA recovery kit.
The vector obtained by tangential restriction of NdeI and XhoI double enzymes was ligated with the PCR amplification product using a seamless cloning kit to construct a recombinant expression vector containing the six ketoreductase polynucleotides of the present invention. The connection is carried out according to the following reaction system: linearized vector pET28a (+) 50ng, primer P1/P2 amplification product 100ng, 2. Mu.L seamless cloning buffer, incubated at 50℃for 20min to obtain recombinant expression vector, PCR verified with T7/T7T universal primer pair (see FIG. 2) and sequencing analysis verified, named pET28a-i after correctness. The recombinant expression vectors pET28a-ii, pET28a-iii, pET28a-iv, pET28a-v, pET28a-vi were obtained in the same manner.
The vector for tangential restriction of NdeI and XhoI double enzymes was ligated with the polynucleotide sequence shown in SEQ ID No.2 using a seamless cloning kit to construct a recombinant expression vector pET28a-0 containing the wild-type ketoreductase polynucleotide of the present invention.
EXAMPLE 4 obtaining recombinant expression transformants
The recombinant expression vectors pET28a-i, pET28a-ii, pET28a-iii, pET28a-iv, pET28a-v, pET28a-vi and pET28a-0 are respectively transformed into BL21 (DE 3) to obtain recombinant expression transformants. The conversion method utilizes a thermal shock method: the competent cells are taken out from a refrigerator at the temperature of minus 80 ℃ and placed on ice, 15min of ice bath is carried out after about 200ng to 500ng of expression vector is added, heat shock is carried out at the temperature of 42 ℃ for 90s, 3min of ice bath is carried out, 500 mu LLB culture solution is added, 220rpm incubation is carried out at the temperature of 37 ℃ for 45min, 50uL is evenly coated on LB solid culture medium containing 50 mu g/ml, the culture is inverted and carried out overnight to obtain monoclonal, 20% final concentration glycerol is used for preservation and minus 80 ℃ after the culture is expanded, and the recombinant expression transformants are named BL21 (DE 3) -i, BL21 (DE 3) -ii, BL21 (DE 3) -iii, BL21 (DE 3) -iv, BL21 (DE 3) -v, BL21 (DE 3) -vi, BL21 (DE 3) -0 after the correct sequencing analysis.
EXAMPLE 5 shake flask culture fermentation of recombinant expression transformants
The recombinant expression transformants (BL 21 (DE 3) -i, BL21 (DE 3) -ii, BL21 (DE 3) -iii, BL21 (DE 3) -iv, BL21 (DE 3) -v, BL21 (DE 3) -vi) obtained in example 4 were inoculated in LB liquid medium (kanamycin was added at 100. Mu.g/ml) and cultured overnight at 37℃and 220rpm, respectively; cultures were transferred at a 1:100 ratio into 50mL fresh LB medium (peptone 10 g.+ -. 0.1 per liter; yeast extract 5 g.+ -. 0.1; naCl 10 g.+ -. 0.1, 250mL shake flask) and grown at 37 ℃. When the optical density at 600nm (OD 600) reached about 0.6, isopropyl thiogalactoside (IPTG) was added to a final concentration of 1mM and the cells were grown at 25℃for 16 hours. Centrifuging at 12000rpm and 4deg.C for 10min, discarding supernatant to obtain cell precipitate as thallus wet cells (whole cells), re-suspending thallus wet cells with pre-cooled 100mM Tris-HCl buffer (pH 7.5) at 200g/L, ultrasonic crushing, centrifuging at 12000rpm and 4deg.C for 30min, collecting supernatant, i.e. crude enzyme solution, and storing at-20deg.C. Shake flask expression polyacrylamide gel electrophoresis, results are shown in fig. 2 and 3. Wherein, the lanes of FIG. 2 are Marker, BL21 (DE 3) -iii protease (the amino acid sequence of which is SEQ ID NO. 5), BL21 (DE 3) -ii protease (the amino acid sequence of which is SEQ ID NO. 4), BL21 (DE 3) -i protease (the amino acid sequence of which is SEQ ID NO. 3), wild type and blank in order from left to right. The lanes of FIG. 3 are, in order from left to right, marker, wild-type, BL21 (DE 3) -iv protease (with the amino acid sequence SEQ ID NO. 6), BL21 (DE 3) -v protease (with the amino acid sequence SEQ ID NO. 7), BL21 (DE 3) -vi protease (with the amino acid sequence SEQ ID NO. 8).
EXAMPLE 6 catalytic hydrogenation of substrate Compounds of formula I by recombinant expression transformants
The cell pellets obtained by centrifugation at 12000rpm at 4℃for 10min using seven recombinant expression transformants obtained in example 5, BL21 (DE 3) -I, BL21 (DE 3) -II, BL21 (DE 3) -iii), BL21 (DE 3) -iv, BL21 (DE 3) -v, BL21 (DE 3) -vi, and wild-type BL21 (DE 3) -0 (Rhodococcus erythropolis) each catalyze the compound of formula I to the compound of formula II in the following manner: 5mL of 0.1M Tris-HCl,500ul NADP+ (20 g/L), 1.0g of an isopropanol solution (containing 5mL of isopropanol) of the substrate (compound represented by formula I), 1.2g of glucose, 0.1g of glucose dehydrogenase (Shang Ke. RTM. Biological medicine (Shanghai), GDH, 40U/mg), 0.4g of wet cell of the cell, were reacted at 40℃at 200rpm, and samples were taken at 10h and 24h, respectively, to determine the conversion of the substrate (HPLC detection of the reaction solution) and the ee value of the product for 24 h. The results are shown in Table 3 below. Under the same expression condition, the high-activity specific enzyme mutants are screened according to different conversion rates and ee values.
Table 3 results of catalytic conversion of substrate (formula I) by wet cells of different expression transformants.
As can be seen from Table 3, the catalytic conversion efficiency of the ketoreductase produced by the wild-type transformant to the compound of formula I was poor, the catalytic conversion efficiency of the ketoreductase produced by BL21 (DE 3) -ii and BL21 (DE 3) -iii) transformants to the compound of formula I was good, the catalytic conversion efficiency of the ketoreductase produced by BL21 (DE 3) -iii) transformant to the compound of formula I was good, and after 20 hours, the substrate conversion rate reached 99.5% and the ee value reached 99.5%.
Example 7: high-density fermentation preparation of recombinant expression transformant BL21 (DE 3) -iii
The recombinant expression transformant BL21 (DE 3) -iii obtained in example 5 was inoculated into 3mL of liquid LB medium, cultured overnight at 37℃under shaking at 220rpm, inoculated into 400mL of liquid LB medium at a ratio of about 1%, and cultured until OD 600 reached 4 as a seed solution, and inoculated into 2L of fermentation medium for high-density fermentation. The initial temperature was 37℃and the stirring speed was 300rpm, aeration rate was 1.5vvm, pH was 6.8, and then the stirring speed was increased continuously up to 1000rpm. The fermentation culture nutrient is in two stages, after inoculation in the first stage, the culture is carried out for about 4 hours, the carbon source consumption in the culture medium is complete, and the feedback feeding is carried out according to DO. The temperature is reduced to 25 ℃ after feeding, dissolved oxygen is kept above 30%, isopropyl thiogalactoside (IPTG) is added for induction after feeding for 8 hours, and the mixture is placed in a tank after 12 hours of induction. The supernatant was centrifuged at 8000rpm for 10min to obtain wet cells (whole cells) of the cells, which were designated as KRED-APTI-100.
Example 8: whole-cell catalytic reduction of KRED-APTI-100 compounds of formula I
50ML of 0.1M Tris-HCl,5.0mL of NADP+ (20 g/L), 10g of an isopropanol solution (volume of isopropanol: 50 mL) of a substrate (compound of formula I), 12.0g of glucose and 0.4g of glucose dehydrogenase (GDH, 40U/mg) were uniformly mixed at room temperature, different amounts of KRED-APTI-100 cell wet cells (used in amounts of 10g/L, 20g/L and 30g/L, respectively) were added thereto, and after uniform mixing, the reaction was performed at 40℃per 200rpm, the pH of the system was controlled at 7.5 (using a 2M aqueous NaOH solution) during the reaction, and samples of the reaction solution were subjected to HPLC analysis (see Table 4 for specific results). As can be seen from Table 4, after 24 hours, the amount of the KRED-APTI-100 cell wet cells can lead the conversion rate of the substrate to reach more than 90 percent; after 36h, the substrate conversion rates of the cell wet cells with the dosage of 20g/L and 30g/L can reach 99.5 percent and 99.7 percent respectively.
The reaction system (the amount of wet cells of the thalli is 20g/L, the reaction solution is reacted for 36 hours) is concentrated to about 1/2 of the original volume, 100mL of isopropanol is added into the reaction system at normal temperature, the mixture is stirred for 30min and then filtered, 100mL of ethyl acetate is added into the filtrate for extraction, and the obtained organic phase is concentrated to obtain 9.8g of crude product containing the compound IV, the yield is 95.0%, the purity is 97.2%, and the ee value is more than 99% after HPLC detection (see figure 3).
Table 4 catalytic reduction conversion data for substrates (compounds of formula I) at various times with varying KRED-APTI-100 cell wet cell usage.
| The dosage of wet cells is 10g/L | Dosage of wet cells 20g/L | The dosage of wet cells is 30g/L | |
| Conversion for 12h | 60.3％ | 75.5％ | 80.6％ |
| 24H conversion | 90.7％ | 95.9％ | 98.1％ |
| 36H conversion | 92.6％ | 99.5％ | 99.7％ |
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A ketoreductase mutant, characterized in that the amino acid sequence of the ketoreductase mutant is an amino acid sequence obtained by mutating the amino acid sequence shown in SEQ ID No.1, wherein the mutation at least comprises one of the following mutation sites:
Mutation from Y to F or M at position 157, from K to M or A at position 161, from P to A or W at position 187, from T to L or A at position 192, or
The ketoreductase mutant has a mutation site in a mutated amino acid sequence, and the amino acid sequence of the ketoreductase mutant has more than 85% homology with the mutated amino acid sequence.
2. The ketoreductase mutant of claim 1, wherein the ketoreductase mutant has an amino acid sequence as shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8, or
The amino acid sequence of the ketoreductase mutant has more than 85% homology with the amino acid sequence shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8.
3. The ketoreductase mutant according to claim 2, characterized in that the amino acid sequence of the ketoreductase mutant is shown as SEQ ID No.4 or SEQ ID No.5, more preferably the amino acid sequence of the ketoreductase mutant is shown as SEQ ID No. 5.
4. A polynucleotide encoding a ketoreductase mutant according to any one of claims 1 to 3.
5. The polynucleotide according to claim 4, wherein the sequence of the polynucleotide is shown as SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO.14, or
The amino acid sequence of the ketoreductase mutant has more than 85% homology with the nucleotide sequence shown as SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO. 14.
6. A recombinant expression vector comprising the polynucleotide of claim 4 or 5.
7. A host cell comprising the recombinant expression vector of claim 6.
8. The use of a ketoreductase mutant according to any one of claims 1 to 3 for catalyzing the preparation of chiral alcohols from ketones,
The structural formula of the ketone compound isWherein R 1 is selected from phenyl, R 2 is selected from C 1～C8 alkyl, wherein said phenyl and said C 1～C8 alkyl are optionally further substituted with one or more substituents selected from hydroxy, amino, halo, nitro, cyano, carboxy, C 1～C8 alkyl, C 1～C8 alkoxy, C 5～C10 cycloalkyl, C 2～C6 ester, C 2～C8 alkenyl, C 2～C8 alkynyl, C 4～C11 heterocyclyl, C 5～C10 aryl, C 5～C10 heteroaryl,
The alcohol compound isWherein R 1 and R 2 are as defined above.
9. The use according to claim 8, wherein the ketone compound is a compound of formula I,
The alcohol compound is a compound of a formula II,
10. A process for the preparation of a compound of formula II, comprising the steps of:
Reducing a compound of formula I in the presence of a ketoreductase to produce a compound of formula II, the reaction being as follows:
wherein the amino acid sequence of the ketoreductase is shown as SEQ ID NO. 5.
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